Induction of allograft tolerance would avoid the need for chronic immunosuppressive therapy with its attendant toxicities. We have recently performed a pilot trial of combined non-myeloabiative bone marrow and kidney transplantation (CKBMT) from related haploidentical donors. Four of 5 patients achieved durable allograft acceptance without maintenance immunosuppression, representing the first successful intentional achievement of tolerance to HLA-mismatched allografts. In vitro studies revealed the development of specific unresponsiveness to the donor in assays that primarily measure direct alloreactivity. Regulatory T cells (Tregs) were enriched in the recovering peripheral blood T cell populations, and suppression of anti-donor reactivity was detected variably within the first year post-transplant. However, early acute humoral rejection episodes occurred in several patients, resulting in the addition of B cell-depleting anti-CD20 mAb to the regimen. Several patients developed autoantibodies and donor-specific alloantibodies in the presence of low T cell counts and/or T cell tolerance in vivo and in vitro. We hypothesize that these antibodies are generated by recovering transitional B cells in the absence of T cell help. However, an alternative hypothesis is that indirectly alloreactive T cells provide help for class-switched Ig responses in these patients, despite the donor unresponsiveness seen in bulk MLR and CML assays. The protocol is now being reopened to include both HLA-haploidentical and more extensively mismatched donors. We propose to address several hypotheses in order to understand the tolerance achieved through CKBMT. We will: 1) Assess tolerance of directly and indirectly donor alloreactive T cells in recipients of HLA-mismatched CKBMT. Indirect responses to both hematopoietic and renal tubular cell-derived antigens will be measured and interpreted in the context of the development of anti-donor antibodies;and 2) Assess the role of regulatory cells, including FoxP3+ Treg and CD127-C025-C04 cells, in the development of donor-specific tolerance in recipients of CKBMT. We will assess the phenotype and function of regulatory cell populations obtained from patient PBMC and expanded from renal biopsy specimens. We hypothesize that donor-specific regulatory cells will be enriched in the renal allograft compared to the PBMC and that donor-specific regulatory capacity will decline over time as deletion of donor-reactive T cells occurs. In combination with studies performed at the ITN core facilities, our studies should promote a comprehensive understanding of the mechanisms involved In tolerance induced by CKBMT.